title: Behavior of Plasmodium falciparum purine nucleoside phosphorylase in macromolecular crowded environment.
abstract: Biochemical and biophysical properties of enzymes have been studied in dilute buffer system, which are far from the crowded physiological condition of cell. We report the enzyme kinetics and refolding of Plasmodium falciparum purine nucleoside phosphorylase under crowded conditions. Enzyme catalytic efficiency was inversely affected in the presence of polyethylene glycols and Dextran whereas it was increased in the presence of osmolytes. We detected a non-linear relationship between Km and increasing macromolecular crowding agents. At low concentrations of PEGs and Dextran, we observed decreased substrate binding whereas higher concentrations of PEGs and Dextran favored substrate binding. The presence of sucrose decreased the Km values. We detected decrease in Kcat value in the presence of PEGs and Dextran, whereas osmolytes increased the Kcat values. Thermal resistance of enzyme was increased in the presence of crowding agents. Intrinsic and extrinsic fluorescence analysis indicated change around active site loop region having single tryptophan residue. Preferential exclusions of polyols favor the catalytic mechanism of the enzyme. Urea denatured enzyme showed fast refolding when diluted and rate of refolding was not affected by the presence of crowding agents. It is important to draw together significant knowledge about modulation of inherent properties of this enzyme in crowded environment which will be helpful in better understanding of this drug-target enzyme and in further inhibitor design.
authors.affiliation: Division of Biochemistry, CSIR-Central Drug Research Institute, Chattar Manzil Palace, Lucknow, UP 226001, India.
authors.forename: Manish Kumar
authors.initials: MK
authors.lastname: Suthar
authors.forename: Pawan Kumar
authors.initials: PK
authors.lastname: Doharey
authors.forename: Anita
authors.initials: A
authors.lastname: Verma
authors.forename: Jitendra Kumar
authors.initials: JK
authors.lastname: Saxena
mesh_terms: D002384:Catalysis / Q000187:drug effects; D003911:Dextrans / Q000494:pharmacology; D006151:Guanosine / Q000737:chemistry; D007700:Kinetics; D046911:Macromolecular Substances / Q000737:chemistry*; D010963:Plasmodium falciparum / Q000201:enzymology*; D011092:Polyethylene Glycols / Q000494:pharmacology; D011489:Protein Denaturation / Q000187:drug effects; D058849:Protein Refolding / Q000187:drug effects; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry*; D013379:Substrate Specificity; D014508:Urea / Q000494:pharmacology
chemical_list: D003911:Dextrans; D046911:Macromolecular Substances; D006151:Guanosine; D011092:Polyethylene Glycols; D014508:Urea; D011683:Purine-Nucleoside Phosphorylase
keywords: Crowding agents; Enzyme kinetics; Fluorescence; Purine nucleoside phosphorylase